Population pharmacokinetic modeling of dolutegravir/lamivudine to support a once-daily fixed-dose combination regimen in virologically suppressed adults living with HIV-1

ABSTRACT A fixed-dose combination (FDC) of 50 mg dolutegravir and 300 mg lamivudine is indicated for the treatment of HIV-1 infection. This analysis aimed to characterize the population pharmacokinetics (PK) of dolutegravir and lamivudine based on data from a phase 3 study (TANGO) in virologically suppressed adults living with HIV-1 switching to dolutegravir/lamivudine FDC. These analyses included 362 participants who contributed 2,629 dolutegravir and 2,611 lamivudine samples collected over 48 weeks. A one-compartment model with first-order absorption and elimination parameterized by apparent oral clearance (CL/F), apparent volume of distribution (V/F), and absorption rate constant (Ka) described dolutegravir PK. Covariate search yielded body weight, bilirubin, and ethnicity as predictors of CL/F, and weight was predictive for V/F. The estimates of CL/F, V/F, and Ka were 0.858 L/h, 16.7 L, and 2.15 h−1, respectively. A two-compartment model with first-order absorption and elimination parameterized by CL/F, apparent intercompartmental clearance (Q/F), apparent central volume of distribution (V2/F), apparent peripheral volume of distribution (V3/F), and Ka described lamivudine PK. Covariate search yielded eGFR and race as predictors of CL/F, and weight was predictive for V2/F. The estimated parameter values were CL/F = 19.6 L/h, Q/F = 2.97 L/h, V2/F = V3/F = 105 L, and Ka = 2.30 h−1. The steady-state prediction suggested that the effect of covariates dolutegravir and lamivudine exposures was small (<20%) and not clinically relevant. Therefore, no dose adjustments are recommended based on these analyses. The results support the use of dolutegravir/lamivudine FDC in the treatment of HIV-1 infection in adults. CLINICAL TRIALS This study is registered with ClinicalTrials.gov as NCT03446573.

A fixed-dose combination (FDC) tablet of 50 mg dolutegravir and 300 mg lamivudine was recently approved by the United States (US) Food and Drug Administration and European Medicines Agency for the treatment of HIV type-1 (HIV-1) infection in adults in the US and adults and adolescents in the European Union (5,6).Dolutegravir/lamivudine 2DR is indicated as a complete regimen for HIV-1 treatment in adults with no prior antiretroviral treatment history and no known substitutions associated with resistance to dolutegravir or lamivudine.Individuals should be tested for hepatitis B infection prior to or when starting therapy.Two phase 3, multicenter, double-blind, randomized, noninferiority studies (GEMINI-1 and GEMINI-2) evaluated the safety and efficacy of dolutegravir/lamivudine single-entity tablets as a complete 2DR for the treatment of HIV-1 infection in therapy-naïve adults compared to a 3DR of dolutegravir/tenofovir disoproxil fumarate/emtricitabine.These studies showed that dolutegravir/lamivudine 2DR was safe, well tolerated, and exhibited long-term noninferior efficacy in achieving virologic suppression compared to the 3DR (7,8).
A separate clinical trial in healthy participants evaluated the bioequivalence in fasted state, food effect, and safety of a fixed-dosed formulation of 50 mg dolutegravir and 300 mg lamivudine compared to the same doses coadministered as single-entity tablets (9).This study showed that the FDC met bioequivalence standards for dolutegra vir and lamivudine area under the concentration-time curve (AUC) and dolutegravir maximum concentration (Cmax).However, lamivudine Cmax was approximately 32% higher when administered with dolutegravir as an FDC compared to coadministration as single entities, suggesting a difference in the rate but not the extent of absorption for lamivudine in the fixed-dose formulation.The food effect for dolutegravir and lamivudine administered as an FDC tablet was found to be comparable to the known effects of food on the single entities.The results from this study supported the use of a fixed-dose formulation and demonstrated equivalency between the FDC tablet and the coadministered, single-entity tablets used in the GEMINI studies (9).
TANGO was a phase 3, randomized, multicenter, parallel-group, noninferiority study evaluating the efficacy, safety, and tolerability of switching to dolutegravir/lamivu dine FDC tablet in HIV-1-infected adults who are virologically suppressed (Fig. 1A).This study compared switching to once-daily dolutegravir/lamivudine FDC from a once-daily tenofovir alafenamide fumarate (TAF)-based regimen.A 48-week primary endpoint analysis of the phase 3 TANGO study showed that dolutegravir/lamivudine was noninferior in maintaining virologic suppression compared to a TAF-based regimen with no reported virologic failure or emergent resistance (10).A pharmacokinetic (PK) substudy in the dolutegravir/lamivudine FDC arm of the TANGO study was conducted to evaluate dolutegravir and lamivudine concentrations using sparse PK sampling collected in all participants and intensive PK sampling collected in a subgroup of participants to provide sufficient data for a thorough characterization of absorption phase (Fig. 1B).The objectives of this PK substudy, as reported herein, were to characterize the dolutegravir and lamivudine steady-state PK following once-daily oral administration of the dolutegravir/lamivudine FDC using population modeling methods to identify important determinants of variability and to provide post hoc steady-state exposure metrics (AUC0-τ, Cmax, and Cτ; τ = 24 hours post-dose) for both analytes.This analy sis supported the regulatory approval of dolutegravir/lamivudine FDC in virologically suppressed adults with HIV-1.

Study design and pharmacokinetic analysis
TANGO was a phase 3, randomized, open-label, noninferiority study designed to determine whether HIV-infected adults with virological suppression on a ≥three-drug TAF remain suppressed after switching to a 2DR of dolutegravir 50 mg/lamivudine 300 mg given once daily (10).Participants were randomized 1:1 to the dolutegravir/ lamivudine regimen or current TAF, and antiviral activity was monitored over 48 weeks.In an intensive PK substudy, serial PK samples were collected in fasted state in a subset of 30 participants receiving dolutegravir/lamivudine at week 4 (pre-dose, 0.5, 1, 1.5, 2, 3, 4, 6, 10, and 24 hours post-dose).Sparse samples were obtained without regard to food from most dolutegravir/lamivudine-assigned participants at weeks 4 (pre-dose and 1 hour post-dose), 8 (1-4 hours or 4-12 hours post-dose), 12 (4-12 hours or 1-4 hours post-dose), and weeks 24, 36, and 48 (pre-dose).Plasma samples were analyzed for dolutegravir and lamivudine concentrations using validated analytical methods based on protein precipitation followed by UHPLC-MS/MS analysis with a TurboIonSpray interface and multiple reaction monitoring.The lower limit of quantification in plasma was 20 ng/mL for dolutegravir and 2.5 ng/mL for lamivudine (9).

Modeling methods
Dolutegravir and lamivudine concentrations were modeled in NONMEM version 7.3 (ICON Development Solutions, Ellicott City, MD) using first-order conditional estimation method with interaction, and run management was conducted with Pirana version 2.9.0 (Certara, L.P., Princeton, USA).Post-processing, diagnostic plots, and covariate analysis were conducted in R version 3.2.5.There were only 0.19% dolutegravir and 0.11% lamivudine samples below limit of quantification (BLQ) in this study.Given the smaller number of samples, BLQ modeling methods were not attempted and excluded from analysis.Dolutegravir model fitting began using a previously described internally developed one-compartment linear model with first-order absorption, parameterized for the absorption rate constant (Ka), apparent clearance (CL/F), apparent volume of distribution (V/F), and absorption lag time (ALAG) (11).Weight, smoking status, age, and total bilirubin were predictors of CL/F, weight was a predictor of V/F, and gender was a predictor of bioavailability.Addition of an ALAG and inter-individual variability (IIV) on Ka were explored in the current model development but were not supported by the data and thus were not retained.Informed by a previous internally developed population PK model for twice-daily dosing of lamivudine (12), base lamivudine model development began with a one-compartment linear model with first-order absorption and weight, and total creatinine clearance (CrCL) was the predictor of CL/F.Various residual error models were also explored in this model development, including proportional, mixed propor tional, and additive error, with or without an IIV term on the proportional residual error.Model selection was guided by goodness-of-fit plots, successful convergence, plausibility and precision of parameter estimates, and objective function value (OFV).Following identification of the base structural models, covariate hypotheses for each analyte were tested.The impact of covariates chosen that could affect dolutegravir [weight, age, sex, smoking status, albumin level, total bilirubin, serum creatinine, estimated glomerular filtration rate (eGFR), Centers for Disease Control and Prevention (CDC) HIV classification (1 vs 2 and 3), ethnicity, race, and concomitant medications] or lamivudine [age, race, sex, serum creatinine, eGFR, CDC HIV classification (1 vs 2 and 3), ethnicity, creatinine clearance, and concomitant medications] PK parameter estimates for CL/F and/or V/F was assessed visually in each drug's respective model with the forward/backward approach.Covariates with a significant impact on estimated CL/F or V/F OFVs were added to base models to become full covariate models.The effect of a covariate was tested by centering at the population median value and using a power model with estimated exponent.Covariates were retained in the final full models if a significant impact on PK parameter OFV was observed after a backward elimination process of the full covariate models.For forward and backward selections, a significance level of 0.01 [corresponding to 6.63 points change in the OFV for 1 degree of freedom (d.f.)] and 0.001 (corresponding to 10.83 points change in the OFV for 1 d.f.), respectively, for covariate model development was used.Predictive performance of final models was evaluated by prediction-corrected visual predictive checks (pcVPCs) and bootstrap analysis.

Simulation methods
The final dolutegravir and lamivudine models were used to compute individual steadystate AUC0-τ, Cmax, and Cτ values for dolutegravir and lamivudine following steadystate dosing of treatment-experienced participants in the TANGO study.Steady-state concentrations were predicted at 0, 1, 2, 3, 4, 6, 8, 12, and 24 hours following a once-daily dose of dolutegravir 50 mg/lamivudine 300 mg.AUC0-τ (linear up/log down trapezoidal rule), Cmax, and Cτ were calculated from predicted concentrations by noncompartmen tal analysis in R software.These post hoc estimates of exposure were divided and summarized by categorical and continuous covariates identified to significantly influence PK or by population quartiles.In addition, in order to predict the impact of covariates on dolutegravir or lamivudine exposure, simulations were performed within NONMEM 1,000 times per subject for a total of 361,000 (361 × 1,000) simulated profiles.All participants from the population PK analysis (361 participants with their own set of covariates) were used for the simulation.The full population was first divided into categories or quartiles for categorical or continuous covariates, respectively.The geometric mean of each trial was computed for each category or quartile and normalized by the median geometric mean of the overall population serving as a reference parameter.

RESULTS
A total of 2,629 dolutegravir and 2,611 lamivudine samples from 362 participants were included in the population PK analysis.Participants were primarily male, White, and below 65 years old.HIV-A was the most common HIV classification in the analysis population.The majority (95%) of participants had a CDC HIV infection classification of 1, 2, or 3 at baseline (Table 1).Concomitant medication use fluctuated by study visit, but only metal cation-containing products (11%-13%) and cytochrome P450 (CYP) 3A inhibitors (17%-19%) were consistently used in >10% of population and included in the

Dolutegravir PK model
The final model selected for dolutegravir was a one-compartment model with first-order absorption and elimination.Residual error was fit to a proportional residual error model, and IIV was included on CL/F and the magnitude of proportional error.Bilirubin and ethnicity decreased OFV by 14.265 and 16.635 points, respectively, and were identified as predictors of dolutegravir CL/F; weight reduced OFV by 79.62 points and was identified as a predictor of both CL/F and V/F (Table 2).These covariates were incorporated into the final dolutegravir PK model to estimate post hoc exposures.In Hispanic or Latino participants, CL/F was 15.6% lower compared to those of non-Hispanic or Latino ethnicity.Over the range of observed bilirubin levels (2-34 µmol/L) in the analysis population, CL/F ranged from 24% higher to 20% lower than that of a typical subject with a bilirubin level of 8 µmol/L.Over the range of weights included in the analysis (50-153 kg), CL/F ranged from 18% lower to 33% higher, and V/F ranged from 34% lower to 83% higher than for a 79-kg subject.IIV on CL/F and the proportional residual error term were moderate at 26.6% and 24.2%, respectively, and all PK parameters were estimated with good precision as evidenced by relative standard error values <25%.Model diagnostics, as evaluated by goodness-of-fit plots (Fig. S1), bootstrap (Table 2), and normal prediction distribution error (NPDE) plots (Fig. S2), suggested adequate model fit and that predictive perform ance was acceptable for simulations.The median tendency of absorption phase was not captured effectively; however, overall, pcVPC plot indicates a good agreement between model predictions and dolutegravir individual observations (Fig. 2A).
Based on the final model, individual post hoc estimates of exposure (AUC0-τ, Cmax, and Cτ) were summarized based on the categorical covariates or by quartiles of exposures.The AUC0-τ, Cmax, and Cτ for individual participants increased with increasing bilirubin concentration, with a difference between quartiles with the lowest and highest bilirubin level groups of 12%, 13%, and 45%, respectively.Similarly, AUC0-τ, Cmax, and Cτ were 17%, 13%, and 35% higher in Hispanic/Latino participants, respec tively, than in non-Hispanic/Latino participants.Participants of higher weight demonstra ted lower predicted AUC0-τ and Cmax, with a difference between quartiles with the highest and lowest weight groups of 20% and 22%, respectively.Cτ values were similar across the weight groups.

Lamivudine PK model
The final once-daily lamivudine model identified was a two-compartment model with first-order absorption and elimination with a proportional residual error model.There was a 126-point decrease in the OFV for the two-compartment model, and the diagnos tic plots displayed a less pronounced bias compared to the one-compartment model , indicated that the 2-CMT model better described the lamivudine data.However, fitting the two-compartment model to the full data set resulted in difficulties estimating reliable values for V3/F.Therefore, model simplification was explored by fixing the V3/F value to either the estimated value from the model fit to the intensive PK subset (59.8 L) or to the value of V2/F in the subsequent runs, respectively.The two methods resulted in similar goodness-of-fit plots.However, V2/F = V3/F resulted in a lower OFV compared to intensive PK subset value by 21.8 points.Therefore, the V3/F = V2/F parameterization was carried forward.The IIV was included on CL/F and V2/F.Body weight was allometrically scaled on CL/F, apparent distributional clearance (Q/F), and V2/F with fixed exponents of 0.75 on CL/F and Q/F and 1.0 on V2/F.The covariates eGFR and race (Black or African American vs all other races com bined) were included in the final lamivudine model to estimate post hoc exposures as predictors of CL/F based on covariate model results (decreased OFV by 20.85 and 13.79 points, respectively).Specifically, over the range of observed eGFR values (44-147 mL/min/1.73m 2 ), CL/F of participants in the analysis population ranged from 35% lower to 24% higher than that of a typical subject with eGFR of 99 mL/min/1.73m 2 , and CL/F of Black or African American participants was 21% lower than those of other races in the study.As compared to PK parameters for a typical 70-kg subject, over the range of observed weights (50-153 kg), CL/F and Q/F ranged from 22% lower to 80% higher in the analysis population, and V2/F or V3/F ranged from 29% lower to 119% higher.
The magnitude of IIV was low moderate on CL/F and moderate on both V/F and the proportional residual error term.Bootstrap values and 95% confidence intervals (CIs) were nearly identical to NONMEM estimates, and all parameters were estimated precisely (Table 2).Diagnostic plots (Fig. S3) demonstrated an adequate fit of the model to lamivudine data, though some bias was present at low concentrations, likely due to many samples having high pre-dose concentrations (~2%) , inconsistent with a pre-dose time of collection (Fig. S3).The reflection of the bias was more in lamivudine compared to dolutegravir model.This slight bias was similarly reflected through overprediction of trough concentrations in the pcVPC (Fig. 2B).The NONMEM and bootstrap estimates and 95% CI were almost identical for all parameters (Table 2), and NPDE plots displayed a close to normal distribution for the prediction error and did not reveal any particular bias in model predictions following once-daily FDC oral administration (Fig. S4).Overall, based on the goodness of fit, as well on the results from the bootstrap, pcVPC, and NPDE, the final model was deemed to have acceptable predictive performance for simulation purposes.
Final model-derived geometric means for the post hoc exposure steady-state parameters AUC0-τ (µg•h/mL) and Cmax (µg/mL) after once-daily dolutegravir/lamivu dine FDC were 59.2 and 5.08 for dolutegravir and 14.1 and 2.50 for lamivudine, respec tively (Table 3).These model-based post hoc PK parameters calculated in the overall population (361) were comparable to those observed in a subset of 30 participants with intensive PK data (Table S2).Thus, despite the slight overprediction of trough concentra tion in lamivudine pcVPC, the impact of this slight model misspecification was minimal on overall AUC0-τ, which is the best predictor of lamivudine antiviral activity.The level of residual unexplained variability (RUV) can be varied between participants due to sometime incorrectly collected dosing histories or compliance between participants.As mentioned above, ~2% of pre-dose observations were slightly higher compared to other pre-dose concentrations in this study and historical studies.This varying RUV magnitude suggests that the collected data from different participants have different information.Instead of completely removing them without any reason, we have added IIV on residual unexplained variability to better optimize both models (13).

Simulations
Forest plots depict the change in simulated dolutegravir exposure for subpopulations of varying weight, bilirubin levels, and ethnicity (Fig. 3A) and simulated lamivudine exposure for subpopulations of varying eGFR, race, and weight (Fig. 3B).Once-daily  dosing of dolutegravir/lamivudine FDC demonstrated <20% magnitude of significant covariate effects compared to reference.

DISCUSSION
In the present study, dolutegravir and lamivudine concentrations from a phase 3 study of once-daily dosing in virologically suppressed HIV-1 participants were modeled, and impactful covariates were identified.Similarly, to previous modeling analyses (11), dolutegravir concentrations were fit to a one-compartment linear model with first-order absorption and elimination.Effects of bilirubin and ethnicity on CL/F and weight on both CL/F and V/F were observed, though all effects were small and therefore considered to be not clinically meaningful.These results are consistent with previous dolutegravir analyses in HIV-infected treatment-naïve and treatment-experienced participants, and support that no dose adjustment of dolutegravir is necessary to account for bilirubin, ethnicity, or weight variability.Additionally, in this analysis, age, race, sex, smoking, albumin, Centers for Disease Control and Prevention HIV classification, serum creatinine, eGFR, alanine transaminase (ALT), aspartate transaminase (AST), prandial status (fasted state vs taken without regard to food), coadministration with CYP and UDP-glucurono syltransferase inhibitors, or metal cation-containing products did not have significant impacts on dolutegravir PK.In this analysis, lamivudine concentrations were best described by a two-compart ment model with first-order absorption and elimination.This differs from previous lamivudine population PK analysis, which utilized a one-compartment model with first-order absorption and elimination (12).Of note, the previous lamivudine model was based on PK samples collected pre-dose and then at 0.5, 1, 2.5, 3.5, and 8 hours post-dose during twice-daily dosing.Therefore, the discrepancy in model structure likely originates from differences in the sampling strategy due to more samples being collected in the absorption, distribution, and elimination phase in the current study of once-daily dosing in relation to previous lamivudine studies.
The current analysis identified effects of renal function (eGFR) and race on CL/F.Renal function (creatinine clearance) was also identified as a predictor for CL/F in a previous analysis in HIV-infected participants (12).However, similarly to the previous analysis, all covariate effects were small and considered not clinically relevant; thus, no dose adjustment of lamivudine is recommended based on mild renal impairment or race.Lastly, weight was also identified as a predictor of CL/F and V/F in earlier analyses, though this effect was fixed to allometric exponents for this analysis.No effects of CDC classification, HCV co-infection, sex, age, ethnicity, smoking, ALT, AST, bilirubin, or prandial status on lamivudine PK were identified.
Modeled post hoc exposure steady-state parameters AUC0-τ (µg•h/mL) and Cmax (µg/mL) had geometric means of 59.2 and 5.08 for dolutegravir and 14.1 and 2.50 for lamivudine, respectively, after once-daily dolutegravir/lamivudine FDC administration.Overall, these parameters were generally consistent with values reported in previous pharmacokinetic studies in HIV-infected adults with the exception of Cmax of dolute gravir which was higher than that observed in historical data from treatment-naïve populations (Table 3) (11,(14)(15)(16)(17)(18).However, this difference is not considered clinically significant because the safety profile of dolutegravir/lamivudine FDC in the TANGO study is consistent with the safety profile of dolutegravir/lamivudine in treatment-naïve patients (7,10).
Limitations of this study include a population consisting primarily of White (80%) and male (93%) participants.Additionally, the data set utilized for simulations was set to record samples at the subject level.This resulted in inclusion of any covariate correla tions within that population; as such, differences between covariate categories may be confounded in this analysis due to imbalance of other covariates within these categories.
Simulations of steady-state dolutegravir and lamivudine exposure following fixed-dose dolutegravir/lamivudine administration demonstrated <20% magnitude of significant covariate effects compared to reference, signifying a lack of clinical

FIG 2
FIG 2 Prediction-corrected VPC plots of the final population pharmacokinetics models [(A) dolutegravir and (B) lamivudine].Prediction-corrected visual predictive check plot.The solid red line represents the median observed plasma concentration, and the semitransparent red field represents a simulation-based 95% confidence interval for the median.The observed 5% and 95% percentiles are presented with dashed red lines, and the 95% confidence intervals for the corresponding model predicted percentiles are shown as semitransparent blue fields.

aFIG 3
FIG 3 Predicted fold-change in steady-state once-daily dolutegravir (A) and lamivudine (B) AUC0-τ, Cmax, and Cτ relative to reference covariate category.Data from 1,000 simulated trials of n = 361 participants/trial separately for each drug.The line of unity (reference line) represents the median of 1,000 geometric means (from 1,000 simulated trials) for the overall population with.The median of geomeans of the overall population was used to normalize the geometric mean [AUC(0-τ), Cmax, Cτ] of each trial for each covariate category.Bars represent the medians, 2.5th, and 97.5th percentiles of the normalized geometric means for each covariate category.The shaded area represents bioequivalence criteria (0.8-1.25).

TABLE 1
Summary of demographics for participants included in dolutegravir and lamivudine population PK analysis a a ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI, body mass index; BSA, body surface area; CDC classification based on description given in https://www.cdc.gov/hiv/statistics/surveillance/terms.html;GFR calculated using CKD-EPI method; CrCL calculated using Cockcroft-Gault equation.b One subject had an incorrect race reported in the eCRF and was excluded from population PK analysis.covariate analysis.The CYP3A4 inhibitors (weak/moderate/strong) were polled together for this analysis.

TABLE 3
Comparison of 50 mg once-daily dolutegravir and 300 mg lamivudine exposure parameters in HIV-infected adults a